In preparing reagents for convenient and efficient testing of clinical biological samples, it is frequently important to obtain dry chemical blends in uniform, discrete amounts. Thus, reagents are normally precisely measured small, quantities of chemicals often with inert excipients. It has long been known that this is conveniently done by dispensing solutions of the chemicals. Unfortunately, liquid solutions of many materials, and in particular, biologically active materials such as antibodies, enzymes, certain proteins, and hydrolyzable materials are not generally stable in solution. Therefore reagents are often provided in dry form to improve stability.
Current technology for producing dry chemical blends involves procedures such as dry blending, spray drying, fluid bed drying and the formation of rigid spheres. All of these procedures, however, have limitations that make them costly, inefficient or difficult to implement. In dry blending technology, it is difficult to obtain homogeneous blends of chemicals that have different densities. Moreover, homogeneity is particularly difficult to achieve when very small amounts of ingredients are mixed with large amounts of others. Once made homogeneous, it is extremely difficult to reproducibly (within 1 percent) dispense small amounts (less than about 10 mg) of the blended chemicals.
Spray drying technology produces more homogeneous blends of chemicals because the reagents are first dissolved in liquid. Using spray drying, however, it is difficult and costly to obtain precisely sized amounts of blended chemicals. As generally practiced, this process yields particles with size distributions having coefficients of weight variation greater than 20 percent. The resulting particles have to be reprocessed to obtain uniform particle sizes. After agglomeration, the particles are generally less soluble than the original spray dried particles. Moreover, these procedures typically use halocarbons cryogenic solutions which are hazardous to the environment and cannot be employed with some reagents such as antibodies since they may inactivate them.
Fluid bed technology relies upon spraying a liquid reagent blend onto a particle and drying the liquid to obtain a particle coated with the blended reagents. Using this procedure, it is difficult to obtain uniformly sized particles and to produce a uniform product.
A commonly used alternative to production of the desired products is tube coating with a solution of the desired materials followed by heat, air or freeze-drying. Unfortunately, many materials that are tube coated bind strongly to the tube surface and will not rapidly redissolve when liquid is added to the tube. This can complicate assays due to the time lag required to achieve complete dissolution. In some cases, the slow and possibly incomplete redissolution will prevent the development of a useful assay.
It has now been discovered that the use of preformed uniform spheres, eliminates the slow dissolution problem and offers great advantages over tube drying techniques.
The preparation of rigid dry, substantially uniform spheres is an attractive alternative for producing dry blends since the spheres are relatively easy to prepare with defined quantities of ingredients, both active and inert. Additionally, they are easy to handle and dissolve readily in body fluids such as urine, blood, serum and plasma.
Heretofore such spheres or beads have been prepared from aqueous solutions containing detergents or surfactants by dropping measured quantities of the solutions into a cryogenic liquid such as nitrogen, collecting the frozen beads that form and thereafter, lyophilizing the beads to remove the moisture. The beads thus formed are useful for some purposes but cannot be employed in biological tests which are adversely affected by the presence of the residual surfactants which are distributed throughout the beads after lyophilization.
For example, the beads are not useful in tests which require whole blood or take place in the presence of intact red blood cells or intact white blood cells since the surfactants cause blood cell lysis and interfere with the test. This problem is especially acute if the test is one which requires visualization or detection of the product in the absence of quenching by hemoglobin, or intact white blood cells such as used in various chemiluminescent assays as described below.
The art has expended much time and effort seeking to produce dry, stable reagents containing exact quantities of selected components useful to analyze biological samples such as blood, plasma, serum, urine and other body fluids. See, for example the following U.S. Pat. Nos. 3,721,725; 4,820,627; 3,932,943; 4,115,537; 4,848,094; 4,755,461; 4,655,047; 4,678,812; and 4,762,857, which relate generally to dry blending spray drying and fluid bed drying.
A more recently issued patent, U.S. Pat. No. 5,413,732 describes and claims a method for providing lyophilized reagent spheres suitable for analysis of biological samples, in particular analysis of blood samples in a centrifugal analyzer.
A particular advantage of the process described is that the spheres are prepared from an aqueous solution. Hence there is no difficulty in insuring that in the final products the dry components are uniformly distributed and in the proper proportions.
The reagent spheres or beads preferred by the process of the patent are defined as comprising the reagents necessary for the proposed tests together with fillers and surfactants. See for example the first paragraph of column 1, column 6, lines 41 through 60 and all of the examples.
There are, as indicated above, biological tests which cannot be conducted in the presence of surfactants. One such test is a test for sepsis and infections described in J. of Immunol. Methods 212 (1996) 169-165; U.S. Pat. No. 5,804,370; U.S. patent application Ser. No. 06/991,230 filed Dec. 16, 1997, U.S. patent application Ser. No. 991,109 filed Dec. 16, 1997 and in U.S. patent application Ser. No. filed 353,189 filed Jul. 14, 1999. The inventions described in these publications are applicable to recognizing infections and sepsis, including its various stages. The crux of the inventions is the formation of an antibody/antigen complex which binds to specific receptors on neutrophils, lymphocytes or monocytes in the presence of complement to elicit an oxidative burst which can be detected as light emission using, for example, a chemiluminescent agent such as luminol or lucigenin. The level of the oxidative burst is related to the level of the antigen.
The tests are applicable to the recognition of a variety of antigens related to infection and sepsis including, for example, microorganisms and their components, including gram positive cell wall constituents and gram negative endotoxin, lipopolysaccharide, lipoteichoic acid, and the inflammatory mediators that appear in circulation as a result of the presence of these components including tumor necrosis factor (TNF), interleukin-1 (IL-1) and other interleukins and cytokines. Other antigens to which the tests are applicable include those related to drugs of abuse, hormones, toxins, therapeutic drugs, markers of cardiac muscle damage, ovulation, pregnancy and other similar tests.
In accordance with the preferred procedure for conducting the tests, the complementary antibody to the antigen or analyte of interest is distributed in one or more spheres or beads such as those which are the subject matter of this invention.
It has been observed that if the spheres contain surfactants, they cannot be used to conduct the tests described since the surfactants interfere with the various reactions necessary to the production of a detectable and measurable light emission.
Still other tests which are not possible in the presence of surfactants is described in U.S. patent application Ser. No. 353,188 filed Jul. 14, 1999 and Ser. No. 353,190 filed Jul. 14, 1999. These tests also are based on antibody/antigen reactions which produce a detectable signal. While the tests are applicable to detecting a variety of antigens, they are particularly suitable to the detection of cardiac analytes such as myoglobin, troponin I, troponin T and CK-MB which are released into the blood from deteriorating cardiac tissue as a result of a tissue damaging event such as angina or myocardial infarction.
Briefly, the tests involve contacting a suspected sample of whole blood, plasma or serum with a detector antibody which is labeled with a detectable label. Gold is the preferred label since it produces a purple color which is visible to the naked eye. If there is a cardiac analyte complementary to the detector antibody present in the sample, the analyte and antibody will react to form a gold labeled antibody/analyte complex which then reacts with a capture antibody to concentrate the complex and increase the visibility of the purple color.
It is preferred to utilize whole blood for conducting the tests rather than either serum or plasma. However, the red blood cells in whole blood interfere with the visual detection of the color produced by the selected label.
The above identified patent applications describe a device for the rapid detection of cardiac analytes using whole blood. Briefly, the device is a hand held device in which a porous membrane is held between upper and lower rigid supports. The membrane is typically nitrocellulose. The supports are plastic such as an acrylic polymer. The supports, may be transparent to permit the user to see the color formed as a result of the reactions talking place. Alternatively, they may be opaque with an opening through which the delectable signal may be observed.
The device is formed with a pathway for the movement of a test sample from the entry port to the capture antibody. The pathway includes channels formed in the support members which are in cooperative contact with other channels formed in the porous membrane.
In one embodiment of the inventions described in the applications, whole blood enters the pathway and passes around a bead containing a labeled detector antibody, dissolves the bead and its components and then flows onto the porous membrane. The whole blood moves through the membrane by capillary action. As it passes through the membrane the red cells are chromatographically separated from the plasma. The plasma then enters a channel in the membrane where the capture antibody is located.
In a positive test utilizing a gold label, the bead containing the antibody dissolves in the whole blood and the labeled antibody/analyte complex forms. The red blood cells are separated as described and the plasma containing the complex, but free of red blood cells, enters the capture channel where the complex reacts with the capture antibody.
The device may be designed with one or more capture channels to detect one or several analytes. If only one capture channel is used, it may contain one or more spaced capture antibodies. Alternatively, there may be one capture antibody in each of two or more capture channels. Thus, the device may be employed to detect each of the analytes mentioned above or any combination of them. It is, of course not limited to the analytes mentioned but may with appropriate complementary antibodies, either monoclonal or polyclonal, be used to detect any of the large number of analytes known to be released by damaged cardiac tissue. Many such analytes are identified in U.S. Pat. No. 5,749,274 issued on May 5, 1998.
As with the sepsis and infection tests mentioned above, the presence of surfactants in the antibody containing bead interferes with the tests for analyzing whole blood for cardiac analytes. In this case, the interference arises because the surfactants cause hemolysis of the red blood cells releasing all of their components including the richly colored hemoglobin. The hemoglobin cannot be separated by the membrane and, as a result, enters the capture channel and obscures the signal.